ARACHIDONIC ACID METABOLISM IN ENDOTHELIAL CELLS AND PLATELETS

The subject of this work is to examine the hypothesis that some sublytic levels of mechanical perturbation of cells can stimulate cell metabolism. As a marker metabolite, we have chosen arachidonic acid. Principal metabolites for platelets include the cyclooxygenase product thromboxane A2(TXA2) and the lipoxygenase product 12-hydroperoxy-eicosatetraenoic acid (12-HPETE). Polymorphonuclear leukocytes (PMNLs) initally produce principally 5-HPETE, somtimes leading to the formation leukotrienes, though many other metabolites of arachidonic acid have been isolated from activated neutrophils. Human umbilical vein endothelial cells utilize arachidonic acid to produce mainly prostaglandin I2(PGI2). All of these metabolites are biologically active and modulate cell function - sometimes in quite contrasting ways. We will show that levels of sublytic mechanical stress exposure can stimulate arachidonic acid metabolism in all three of the cell types mentioned above. The biological implications of this stress/metabolism coupling may be quite far reaching.Human platelets, leukocytes and endothelial cells all appear to be sensitive to mechanical stress induced activation of arachidonic acid metabolism. Sheared PRP exhibited greatly increased synthesis of 12-HETE and surprisingly little thromboxane B2 production. This indicates that shear stress stimulation of platelets may produce quite different arachidonic acid metabolism than that seen with many direct chemical stimuli, such as thrombin or collagen.Our data demonstrate that a substance derived from shear induced platelet activation may activate the C-5 lipoxygenase of human PMNL under stress, leading to the production of LTB4. We hypothesize that this substance maybe 12-HPETE. LTB4 is known to be a very potent chemotactic factor and to induce PMNL aggregation and degranulation. Our studies provide further evidence that lipoxygenase products of one cell type can modulate production of lipoxygenase products in a second cell type, and that shear stress can initiate cell activation. This kind of coupling could have far reaching implications in terms of our understanding of cell/cell interaction in flowing systems, such as acute inflammation, artificial organ implantation and tumor metastasis.The data on PGI2 production by endothelial cells demonstrate that physiological levels of shear stress can dramatically increase arachidonic acid metabolism. Step increases in shear stress lead to a burst in production of PGI2 which decayed to a steady state value in several minutes. This longer term stimulation of prostacyclin production rate increased linearly with shear stress over the range of 0-24 dynes/cm2. In addition, pulsatile flow of physiological frequency and amplitude caused approximately 2.4 times the PGI2 production rate as steady flow with the same mean stress. Although only PGI2 was measured, it is likely that other arachidonic acid metabolites of endothelial cells are also affected by shear stress.The ability of cells to respond to external stimuli involves the transduction of a signal across the plasma membrane. One such external stimulus appears to be fluid shear stress. Steady shear flow induces cell rotation in suspended cells, leading to a periodic membrane loading, with the peak stress proportional to the bulk shear stress. On anchorage-dependent cells, such as endothelial cells, steady shear stress may act by amplifying the natural thermal or Brownian fluttering or rippling of the membrane. There are several possible mechanisms by which shear stress induced membrane perturbation could mimic a hormone/receptor interaction, leading to increased intracellular metabolism. Shear stress may induce increased phospholipase C activity, caused by translocation of the enzyme, increased substrate (arachidonic acid) pool availability to phospholipase C (particularly from that stored in phosphoinositols) due to shear-induced membrane movements or changes in membrane fluidity, direct activation of calcium - activated phospholipase A2 by increased membrane calcium ion permeability, or most probably by a combination of these mechanisms.

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Arachidonic acid metabolism in platelets and endothelial cells

Progress in Lipid Research ◽

10.1016/0163-7827(81)90076-x ◽

1981 ◽

Vol 20 ◽

pp. 431-434 ◽

Cited By ~ 2

Author(s):

A. Marcus ◽

B. Weksler ◽

E. Jaffe ◽

L. Safier ◽

H. Ullman ◽

...

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Acid Metabolism ◽

Arachidonic Acid Metabolism

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Arachidonic acid metabolism and the adhesion of human polymorphonuclear leukocytes to cultured vascular endothelial cells

Blood ◽

10.1182/blood.v62.4.889.889 ◽

1983 ◽

Vol 62 (4) ◽

pp. 889-895 ◽

Cited By ~ 35

Author(s):

MR Buchanan ◽

MJ Vazquez ◽

MA Jr Gimbrone

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Endothelial Cell ◽

Polymorphonuclear Leukocytes ◽

Acid Metabolism ◽

Arachidonic Acid Metabolism ◽

Vascular Endothelial ◽

Endothelial Monolayers

Abstract Polymorphonuclear leukocytes (PMN) adhere to the vascular endothelial lining in vivo and to the surfaces of cultured endothelial cells in vitro, but the mechanisms of these cellular interactions remain unclear. Arachidonic acid metabolites, both cyclooxygenase- and lipoxygenase-derived, have been shown to influence PMN locomotion, secretion, and adhesion to artificial surfaces. To determine whether such mediators also are involved in regulating PMN-endothelial cell interactions, we have examined the effects of prostacyclin and various inhibitors of arachidonic acid metabolism on the adherence of radiolabeled PMN to cultured bovine aortic endothelial cells. Confluent endothelial monolayers were incubated with washed suspensions of radiolabeled human PMN (which contained less than 1% platelet contamination) at 37 degrees C for 30 min, then subjected to a standardized wash procedure and the number of adherent leukocytes determined radiometrically. Under basal conditions, i.e., in the absence of exogenous activating stimuli, 4,163 +/- 545 PMN adhered per square millimeter of endothelial surface (mean +/- SEM, n = 12). This basal adhesion (which corresponds to approximately 4–5 leukocytes per endothelial cell) was unaffected when the leukocytes and endothelial monolayers were pretreated with cyclooxygenase inhibitors (100 microM aspirin or 1–5 microM indomethacin) or PGI2 (10(-9)-10(6) M). Thus, basal PMN-endothelial adhesion in this in vitro model system does not appear to be dependent on endogenous cyclooxygenase derivatives of arachidonate or to be sensitive to inhibition by exogenous prostacyclin. In contrast, leukocyte adhesion was significantly reduced by pretreatment with 5,8,11,14- or 4,7,10,13-eicosatetraynoic acid, 0.5- 5 mM sodium salicylate, or 10–1,000 microM indomethacin, antiinflammatory agents that can interfere with the metabolism of arachidonic acid via non-cyclooxygenase-dependent mechanisms. These observations may be relevant to the interactions of circulating PMN with vascular endothelium under both physiologic and pathophysiologic conditions in vivo.

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The effect of conjugated linoleic acid on arachidonic acid metabolism and eicosanoid production in human saphenous vein endothelial cells

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids ◽

10.1016/s1388-1981(01)00198-6 ◽

2002 ◽

Vol 1580 (2-3) ◽

pp. 150-160 ◽

Cited By ~ 50

Author(s):

P Urquhart

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Linoleic Acid ◽

Conjugated Linoleic Acid ◽

Saphenous Vein ◽

Acid Metabolism ◽

Arachidonic Acid Metabolism ◽

Human Saphenous Vein ◽

Eicosanoid Production

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The role of endothelial cells in the relaxation of cerebral arteries and arachidonic acid metabolism.

Neurologia medico-chirurgica ◽

10.2176/nmc.27.1152 ◽

1987 ◽

Vol 27 (12) ◽

pp. 1152-1157

Author(s):

Kenji KANAMARU ◽

Shiro WAGA ◽

Tadashi KOJIMA ◽

Kiyoshige FUJIMOTO ◽

Hiroji ITOH

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Acid Metabolism ◽

Cerebral Arteries ◽

Arachidonic Acid Metabolism

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Measurement Of 1-14C Arachidonic Acid Metabolism By Cultured Human Vascular Endothelial Cells And Platelets

10.1055/s-0038-1652875 ◽

1981 ◽

Author(s):

Ch Willems ◽

P J Laanen ◽

G A Pool ◽

Ph G de Groot ◽

J A van Mourik ◽

...

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Acid Metabolism ◽

Vascular Endothelial Cells ◽

Lipid Fraction ◽

Relative Sensitivity ◽

Arachidonic Acid Metabolism ◽

Vascular Endothelial ◽

Response Curves ◽

Stimulation Of

To investigate the relative sensitivity of endothelial cells and platelets towards aspirin (ASA), a method was designed which allows the measurement of prostaglandin (PG) metabolism using [l-14C] arachidonic acid (AA) as precursor. When HEC were incubated with 20 μM AA for 24h at 37°C in the presence of serum, about 15-20% of the label was incorporated into phospholipids whereas 3-4% was present in the neutral lipid fraction. No PG formation could be measured, even after stimulation of the phospholipase system by thrombin or ionophore A 23187. When HEC were incubated for 20 min with AA in the absence of serum, about 8-10% of the label was incorporated into PGs (6-keto PGF1α, 2%; PGE2, 1%; PGF2α, 4%). Platelets were prelabeled with AA, in buffer and the phospholipase system could be stimulated by thrombin (measured as the formation of TXB2, HHT and HETE). Using the above methods, the IC50 of ASA (30 min, 37°C) was 4.7±2.4μM for HEC and 10.1±2.4μM for platelets (p>0.1). Preincubation with 5μM ASA resulted in 50% inhibition of PG synthesis after 30±8 min with HEC and after 40±5.6 min with platelets (p#x003E;0.1). Moreoever when HEC and platelets were combined, incubated with ASA and subsequently tested separately no difference in dose-response curves could be demonstrated. These results indicate that the sensitivity of HEC and platelets to ASA is similar.

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Endotoxin alters arachidonate metabolism in pulmonary endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1987.253.3.c384 ◽

1987 ◽

Vol 253 (3) ◽

pp. C384-C390 ◽

Cited By ~ 25

Author(s):

N. Suttorp ◽

C. Galanos ◽

H. Neuhof

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Lipid A ◽

Acid Metabolism ◽

Cell Damage ◽

Arachidonic Acid Metabolism ◽

Pulmonary Endothelial Cells ◽

Arachidonate Metabolism ◽

Protein And Rna Synthesis ◽

Prostacyclin Synthase

Endotoxin and lipid A dose dependently (1 ng/ml to 10 micrograms/ml) and time dependently (6-24 h) stimulated the generation of large amounts of prostacyclin in cultured pig pulmonary artery endothelial cells. This effect occurred in the absence of cell detachment and overt cell damage. The presence of at least 1% serum was required but the activation of the complement cascade was not. Endotoxin-treated endothelial cells generated increased amounts of prostacyclin upon stimulation with A23187 and arachidonic acid. Endotoxin-induced activation of arachidonate metabolism could be reduced by 10(-10) M glucocorticoids but not by progesterone. It was further affected by inhibitors of protein and RNA synthesis and calmodulin function. In addition, exposure of endothelial cells to endotoxin resulted in an enhanced synthesis of cyclooxygenase and in a higher enzymatic capacity of prostacyclin synthase. The data indicate that endotoxin in concentrations occurring in the plasma of patients profoundly alters arachidonic acid metabolism in endothelial cells.

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